Natural Albino Mutant of Daylily (Hemerocallis spp.) Reveals a Link between Drought Sensitivity and Photosynthetic Pigments Metabolism.

BACKGROUND: Mutant analysis remains one of the main genetic tools for characterising unclarified gene functions in plants, especially in non-model plants. Daylily (Hemerocallis spp.) is a popular perennial ornamental plant grown worldwide. Analysis of daylily mutants can enhance understanding of genes regulating the albino phenotype and improve the cultivar quality of daylily. METHODS: The natural albino mutant (Alb-⁣/-) was isolated by screening a self-pollinated progeny of daylily cultivar 'black-eyed stella'. Transmission electron microscopy was used in analysing the structure of plastids between mutant and wild-type seedlings. The content of chlorophyll, carotenoids and chlorophyll precursors in plants was measured by ultraviolet spectrophotometry. RNA sequencing and physiological measurements were performed to explore the association between drought tolerance and mutation. RESULTS: All the seedlings of the daylily albino mutants died spontaneously within fifteen days after germination when grown in soil. The carotenoid and chlorophyll content in the leaves of the mutant plants significantly decreased compared with those of the wild-type control. The mutant plants displayed stunted growth, and their leaves were white or light yellow in color. Abnormal plastids such as those showing endomembrane vesiculation and lacking stacking were discovered in the leaves of mutant plants. Furthermore, genetic analysis revealed that a single recessive nuclear gene mutation led to the albino trait, RNA sequencing and real-time quantitative PCR validation showed extensive differences in gene expression between the mutant plants and the wild-type control, and most of the genes related to chlorophyll metabolism were down-regulated, with foldchange ranging from 0.20-0.49. Additionally, the surviving homozygous plants (Alb+⁣/+), which do not contain this mutation, were also isolated by analysing the phenotype of their self-pollinated progeny. The net photosynthesis rate and light saturation point of Alb+⁣/+ were higher than those of heterozygous (Alb+⁣/-) plants. Additionally, the Alb+⁣/+ plants were more tolerant to drought conditions than the Alb+⁣/- plants, suggesting that a heterozygous Alb- mutation is sufficient to negatively affect photosynthetic efficiency and drought tolerance. CONCLUSIONS: The albino mutation negatively affects photosynthetic efficiency and drought tolerance, and homozygous mutation is required for the characteristic albino phenotype. This work highlights the link between albino mutation, photosynthetic pigment metabolism and drought sensitivity in daylily.

Genome-wide identification and expression analysis of the SWEET gene family in daylily (Hemerocallis fulva) and functional analysis of HfSWEET17 in response to cold stress.

BACKGROUND: The Sugars Will Eventually be Exported Transporters (SWEETs) are a newly discovered family of sugar transporters whose members exist in a variety of organisms and are highly conserved. SWEETs have been reported to be involved in the growth and development of many plants, but little is known about SWEETs in daylily (Hemerocallis fulva), an important perennial ornamental flower. RESULTS: In this study, 19 daylily SWEETs were identified and named based on their homologous genes in Arabidopsis and rice. Phylogenetic analysis classified these HfSWEETs into four clades (Clades I to IV). The conserved motifs and gene structures showed that the HfSWEETs were very conservative during evolution. Chromosomal localization and synteny analysis found that HfSWEETs were unevenly distributed on 11 chromosomes, and there were five pairs of segmentally duplicated events and one pair of tandem duplication events. The expression patterns of the 19 HfSWEETs showed that the expression patterns of most HfSWEETs in different tissues were related to corresponding clades, and most HfSWEETs were up-regulated under low temperatures. Furthermore, HfSWEET17 was overexpressed in tobacco, and the cold resistance of transgenic plants was much higher than that of wild-type tobacco. CONCLUSION: This study identified the SWEET gene family in daylily at the genome-wide level. Most of the 19 HfSWEETs were expressed differently in different tissues and under low temperatures. Overexpression further suggests that HfSWEET17 participates in daylily low-temperature response. The results of this study provide a basis for further functional analysis of the SWEET family in daylily.

Ethylene Response of Plum ACC Synthase 1 (ACS1) Promoter is Mediated through the Binding Site of Abscisic Acid Insensitive 5 (ABI5).

The enzyme 1-amino-cyclopropane-1-carboxylic acid synthase (ACS) participates in the ethylene biosynthesis pathways and it is tightly regulated transcriptionally and post-translationally. Notwithstanding its major role in climacteric fruit ripening, the transcriptional regulation of ACS during ripening is not fully understood. We studied fruit ripening in two Japanese plum cultivars, the climacteric Santa Rosa (SR) and its non-climacteric bud sport mutant, Sweet Miriam (SM). As the two cultivars show considerable difference in ACS expression, they provide a good system for the study of the transcriptional regulation of the gene. To investigate the differential transcriptional regulation of ACS1 genes in the SR and SM, their promoter region, which showed only minor sequence differences, was isolated and used to identify the binding of transcription factors interacting with specific ACS1 cis-acting elements. Three transcription factors (TFs), abscisic acid-insensitive 5 (ABI5), GLABRA 2 (GL2), and TCP2, showed specific binding to the ACS1 promoter. Synthetic DNA fragments containing multiple cis-acting elements of these TFs fused to ß-glucuronidase (GUS), showed the ABI5 binding site mediated ethylene and abscisic acid (ABA) responses of the promoter. While TCP2 and GL2 showed constant and similar expression levels in SM and SR fruit during ripening, ABI5 expression in SM fruits was lower than in SR fruits during advanced fruit ripening states. Overall, the work demonstrates the complex transcriptional regulation of ACS1.

DhEFL2, 3 and 4, the three EARLY FLOWERING4-like genes in a Doritaenopsis hybrid regulate floral transition.

KEY MESSAGE: DhEFL2, 3 and 4 regulate the flowering of Doritaenopsis . These genes could rescue elf4-1 phenotype in Arabidopsis while its overexpression delayed flowering. Phalaenopsis are popular floral plants, and studies on orchid flowering genes could help develop off-season cultivars. Early flowering 4 (ELF4) of A. thaliana has been shown to be involved in photoperiod perception and circadian regulation. We isolated two members of the ELF4 family from Doritaenopsis hybrid (Doritaenopsis 'Tinny Tender' (Doritaenopsis Happy Smile × Happy Valentine)), namely, DhEFL2 and DhEFL3 (DhEFL4 has been previously cloned). Multiple alignment analysis of the deduced amino acid sequences of the three DhEFL homologs showed that DhEFL4 and DhEFL2 are similar with 72% identical amino acids, whereas DhEFL3 is divergent with 72% similarity with DhEFL2 and 68% similarity with DhEFL4. DhEFL3 forms a separate phylogenetic subgroup and is far away from DhEFL2 and DhEFL4. The diurnal expression patterns of DhEFL2, 3, and 4 are similar in the long-day photoperiod conditions; however, in the short-day conditions, DhEFL3 is different from DhEFL2 and 4. For the DhEFL2, 3, and 4 genes, the strongest audience expression organs are the stem, petal and bud, respectively. The ectopic expression of DhEFL2, 3, or 4 in transgenic A. thaliana plants (Ws-2 ecotype) showed novel phenotypes by late flowering and more rosette leaves. The ectopic expression of DhEFL2, 3, or 4 could complement the elf4-1 flowering time and hypocotyl length defects in transgenic A. thaliana elf4-1 mutant plants. These results strongly suggest that DhEFL2, 3, and 4 may be involved in regulation of flower formation and floral induction in Doritaenopsis.

[Optimizing of cDNA preparation for next generation sequencing].

Next generation sequencing has already been used for genomic analysis of microorganis, human being, animals, and plants. Sample preparation is prerequisite and most important for large-scale sequencing. There are two major interferences for large-scale sequencing, polyA and abundant genes' concealment for rare genes. In order to solve these problems, we used total RNA extracted from violaceae leaves to produce double stranded cDNA. DSN nuclease was used to treat the ds cDNA prior to removing the polyA. Randomly sequencing 100 clones of the treated cDNA showed that there were 94 independent clones in the treated sample, and the sequences did not contained polyA. However, only 62 independent clones were found in the untreated sample, and 15 of the sequencing files were affected by polyA. By randomly sequencing of the treated cDNA, we also found two clones encoded two interested genes. We failed to isolate these genes although the protein mass peaks of them had been found in the MALDI-TOF trace. Furthermore, we designed primers from two known genes with different expression abundances. The PCR yields were approaching similar using the treated cDNAs as templates. These results showed that, removal of the polyA and enrichment of rare genes with DSN can meet the requirements of large-scale sequencing and discovery of new genes.

Identification of candidates for cyclotide biosynthesis and cyclisation by expressed sequence tag analysis of Oldenlandia affinis.

BACKGROUND: Cyclotides are a family of circular peptides that exhibit a range of biological activities, including anti-bacterial, cytotoxic, anti-HIV activities, and are proposed to function in plant defence. Their high stability has motivated their development as scaffolds for the stabilisation of peptide drugs. Oldenlandia affinis is a member of the Rubiaceae (coffee) family from which 18 cyclotides have been sequenced to date, but the details of their processing from precursor proteins have only begun to be elucidated. To increase the speed at which genes involved in cyclotide biosynthesis and processing are being discovered, an expressed sequence tag (EST) project was initiated to survey the transcript profile of O. affinis and to propose some future directions of research on in vivo protein cyclisation. RESULTS: Using flow cytometry the holoploid genome size (1C-value) of O. affinis was estimated to be 4,210 - 4,284 Mbp, one of the largest genomes of the Rubiaceae family. High-quality ESTs were identified, 1,117 in total, from leaf cDNAs and assembled into 502 contigs, comprising 202 consensus sequences and 300 singletons. ESTs encoding the cyclotide precursors for kalata B1 (Oak1) and kalata B2 (Oak4) were among the 20 most abundant ESTs. In total, 31 ESTs encoded cyclotide precursors, representing a distinct commitment of 2.8% of the O. affinis transcriptome to cyclotide biosynthesis. The high expression levels of cyclotide precursor transcripts are consistent with the abundance of mature cyclic peptides in O. affinis. A new cyclotide precursor named Oak5 was isolated and represents the first cDNA for the bracelet class of cyclotides in O. affinis. Clones encoding enzymes potentially involved in processing cyclotides were also identified and include enzymes involved in oxidative folding and proteolytic processing. CONCLUSION: The EST library generated in this study provides a valuable resource for the study of the cyclisation of plant peptides. Further analysis of the candidates for cyclotide processing discovered in this work will increase our understanding and aid in reconstructing cyclotide production using transgenic systems and will benefit their development in pharmaceutical applications and insect-resistant crop plants.

[Functional analysis of specific promoter using vecotors harboring GFP/RFP double fluorescent marker genes].

Most studies related to determining the expression profile of genes and specific promoters used histochemical localization of the reporter gene, gusA. While the histochemical method for visualizing gusA expression suffers from several limitations in the determination of gene expression and location, especially in the tissues with high background acitivty. To solve this problem, a transient expession vector pBI221-RFP/GFP, was constructed using GFP and RFP as double fluorescent marker genes. This vector used CaMV 35S promoter to drive GFP and determine the transforming efficiency. It analyzed expression profile of the target gene and promoter through the RFP activities of the tranformed tissues. Through the specific promoter AGPL1 from watermelon and E8 promoter from tomato, it is resistible to use this vector to study the expression patterns of promoters. Results indicated that the pBI221-RFP/GFP is a very efficient transient expression vector that can be verify the functions of the genes and promoters.

[Carbohydrate metabolism during fruit development of bayberry (Myrica rubra Sieb. et Zucc.)].

The dynamics of dry and fresh weight, the glucose, fructose, sucrose, titratable acid contents, and activities of sucrose-metabolizing and hexose-metabolizing enzymes were examined in developing fruits of bayberry (Myrica rubra Sieb. et Zucc. cvs. 'Wuzi' and 'Biqi'). The results showed the dry and fresh weight of bayberry fruit increased with fruit development and maturation (Fig. 1), with the highest increase rate of dry matters and water occurring during later stage of fruit development (about 10 d before maturation). The change in titratable acid followed a course of "low-high-low" in developing bayberry fruits (Fig. 3). The titratable acid content reached its peak at about 18 d before fruit maturation, and then decreased rapidly. The sugar compositions in fruits of bayberry cv. 'Wuzi' were different from those in fruits of bayberry cv. 'Biqi'. The main sugar accumulated in fruits of bayberry cv. 'Wuzi' was sucrose, accounting for 2/3 of total sugars but the sucrose content in fruits of bayberry cv. 'Biqi' was below 50% of total sugars. The fructose content in fruits of bayberry cv. 'Wuzi' was 4% higher, but that in fruits of bayberry cv. 'Biqi' was 12% lower than glucose content (Fig. 2). The activities of sucrose cleavage enzymes (invertase and cleavage activity of SS) in the fruit of bayberry cv. 'Biqi' increased with fruit development and maturation, but those activities in fruit bayberry cv. 'Wuzi' were almost stable during fruit development with lower levels of enzyme activities in fruit of cv. 'Wuzi' than in cv. 'Biqi' throughout fruit development (Fig. 4 and Fig. 5A). The SPS activity increased during fruit development (Fig. 6), however, the activity peak of synthetic activity of SS occurred at the middle stage of fruit development (Fig. 5B). The FRK activity in fruit of bayberry cv. 'Wuzi' was higher than that of HXK, but the reverse was in fruit of bayberry cv. 'Biqi' (Fig. 7). These results suggested that the 2-3 weeks before fruit maturation was a key phase for the bayberry development and the formation of fruit quality. There was a correlation between water transport and dry matter accumulation. The different sucrose constitutions between two varieties may be attributed to the differences in the activity levels of the sucrose cleavage enzymes while the difference in the ratio of glucose content to fructose content may be caused by the different activity levels of the hexose-metabolizing enzymes.

[Genetic transformation of peach immature cotyledons with its antisenes ACO gene].

Genetic transformation of peach immature cotyledons with its ACO antisense gene was studied by using particle bombardment method through Agrobacterium tumefaciens. Kanr shoots and Kanr plantlet were obtained. The plantet with ACO antisenes gene through Agrobacterium tumefaciens was obtained by micrografting technique and survived for nearly one month. The results of the PCR, PCR-southern, genomes southern hybridization analysis and GUS color reaction of some Kanr materials showed in some degree that the peach ACO antisens gene was integrated into peach genomes.

[The relationship of fructokinase and sugar accumulation during fruit development in satsuma mandarin].

Sugars accumulation and fructokinase activity during satsuma mandarin fruit development in relation to the effect of extra nitrogenous fertilizer on the activity and expression of fructokinase were studied. The results exhibited that fructokinase activity in the tissues of edible and peel decreased during fruit development, which coincided with the accumulation of sugars, while the contents of sucrose and glucose decreased, and the activity of the enzyme increased in peel tissues of ripened fruit. After fertilizing with extra urea, the ratios of sucrose and fructose decreased in ripe fruit, while that of glucose increased compared to the control. The activity of fructokinase presented on a protein basis increased in treated fruit. Northern analysis confirmed that extra nitrogenous fertilizer enhanced the expression of Cufrk1 at the late stage of fruit development, but had no effect on Cufrk2. The results suggest that the two different genes of citrus FRK may play distinct roles in sink metabolism and Cufrk1-encoded fructokinase protein could be induced by fertilization with extra nitrogen.